The overall objective of the proposed research is to gain insight into the biochemistry and physiological role of human beta-carotene 15,15'-dioxygenase (BCDO), an enzyme that catalyzes the first step in the synthesis of vitamin A from dietary provitamin A carotenoids. This pathway is especially crucial for persons on a vegetarian diet lacking preformed vitamin A. Derivatives of vitamin A serve as ligands for the retinoic acid nuclear hormone receptors that are essential for development and normal physiological functions. We have isolated and characterized the cDNA and gene that encode the human BCDO enzyme, as well as initiated biochemical characterizations of the purified recombinant enzyme. Our findings raise a number of questions relating to the physiological roles of BCDO in different tissues, and how the enzyme's biochemical properties influence these roles. Our initial goal will be a detailed analysis of tissue distribution and cell type-specific expression of the BCDO enzyme. These analyses will be accomplished using immunoblotting, immunohistochemistry, RNA blotting, in situ mRNA hybridization, and BCDO enzyme assays. Our second objective is to identify potential subjects with vitamin A deficiency-like symptoms. If these symptoms are a result of mutations in the BCDO gene, we will analyze how the mutations cause biochemical abnormalities in the enzyme function. These studies will help to define important structural domains of the protein. The third goal is to expand the structure-function information derived from naturally occurring mutations by creating, and expressing additional site-directed mutations and domain chimeras in the BCDO using the baculovirus system. We will focus on identifying amino acids that form substrate and nonheme iron binding domains of the protein. Detailed kinetic, pharmacological, and structural properties of the purified mutant enzymes will be compared to those of the wild-type enzyme. The final objective is to identify accessory/activating proteins of BCDO by use of two distinct expression cloning strategies, along with a proteomics approach that involves mass spectrometry of immunoprecipitated BCDO from human tissue extracts. Identified proteins will be cloned, expressed, purified, and tested in a reconstituted system together with recombinant BCDO. The Michaelis-Menten constants of the BCDO enzyme, alone or complexed with selected proteins, will be determined, as well as the mechanism by which the accessory/activating protein(s) act. The studies described in this proposal will further our knowledge on vitamin A synthesis from dietary provitamin A carotenoids in man.